The immune system of mammals has evolved different strategies to defend the organism against the variety of potentially infectious agents. The ability to acquire specific and anamnestic responses against intruders features the adaptive immune system. Main players in the adaptive immune system are B and T lymphocytes, and the specific recognition of antigen by these cells is mediated by receptors with some degree of structural similarity, yet functionally very different. The different receptor specificities are made possible through somatic rearrangement of a limited number of genes and are clonally distributed. The main strategy of this system is to generate a nearly unlimited number of specificities to cover the recognition of almost any foreign antigen. Immunological memory is partly a result of clonal expansion of subsets of T and B cells reacting with a particular antigen, and enables the organism to respond more quickly at the second encounter with the same anti gen.
Cell proliferation is used as a parameter for immune activation. According to the clonal selection theory, exposure to antigen leads to activation of individual B and T cell clones with corresponding receptor-specificities. However, the number of cells with affinity for a certain antigen is a small fraction of the total number of cells (˜0,001%). It is therefore crucial that the activated cells are capable of proliferation (clonal expansion) in order to generate an adequate immune response. Thus, proliferation is a very important parameter characterizing lymphocyte function and capability of immune activation. In in vitro experiments, it is possible to activate the total population of isolated T lymphocytes by using antibodies directed against the antigen receptor complex (TCR/CD3). This will mimic the in vivo situation when T cells are immunoactivated to clonal expansion through the antigen receptor. It is known that T cell proliferation is inhibited through the cAMP signalling, pathway.
Cyclic AMP-dependent protein kinase (PKA) is an enzyme present in all cells. Hormones and neurotransmitters binding to specific receptors stimulate the generation of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP). Cyclic AMP is one of the most common and versatile second messengers and exerts its action by binding to and activate PKA. PKA is a serine/threonine protein kinase which phosphorylates a number of different proteins within a cell, and thereby regulates their activity. It is known that PKA regulates a vast variety of cellular processes such as metabolism, proliferation, differentiation and regulation of gene transcription. PKA is made up of four different subunits, a regulatory (R) subunit dimer and two catalytic (C) subunits. Furthermore, two main classes of PKA isozymes, PKA type I and PKA type II (PKAI and PKAII, respectively) have been described. PKAI and PKAII can be distinguished due to their R subunits, designated RI and RII. Isoforms of RI and RII are called RIα, RIβ, RIIα, RIIβ. Moreover, the C subunits also exist as isoforms referred to as Cα, Cβ and Cγ. The different subunits may form multiple forms of PKA (isozymes) potentially more than 18 different functions.
PKA is a key negative regulator of lymphocyte function. The present inventors and other have shown that cAMP inhibits T lymphocyte proliferation induced through the T cell antigen receptor/CD3 complex (TCR/CD3). We have shown that T cells express both PKAI and PKAII. However, only the selective activation of PKAI is sufficient to mediate the inhibitory effect of cAMP. In addition we have demonstrated that PKAI, but not PKAII, redistribute to, colocalize with and inhibit signalling through antigen receptors on T and B cells and natural killer cells and regulate mitogenic responses in T and B cells and acute cytotoxic responses in NK cells. Thus, PKAI serve as a key negative regulator of lymphocyte functions e.g. mitogenic and cytotoxic responses initiated through antigen receptors.
HIV and Common variable immunodeficiency (CVI). Both primary and secondary immunodeficiencies cause an increased incidence of opportunistic infections and cancer, and are increasing causes of morbidity and mortality in all parts of the world. Human immunodeficiency virus (HIV) causes a chronic infection leading to severe dysfunction of the immune system with markedly increased incidence of a large number of infections and certain forms of malignancies (e.g. lymphoma and Kaposis' sarcoma). In many communities in USA, HIV infection is the leading cause of death among “young” adults. In the developing world this problem is even larger. Next to immunoglobulin (Ig) A deficiency, common variable immunodeficiency (CVI) is the most frequent type of primary immunodeficiency. This form of primary hypogammaglobulinaemia is characterized by onset of immunodeficiency after the first two years of life, by severely decreased serum IgG level and recurrent bacterial infections, particularly in the respiratory tract.
Cellular defects in immunodeficiencies. T cell dysfunction is the immunologic hallmark of HIV infection. Defective lymphocyte cytokine production and impaired proliferative response on stimulation are early signs of immunodeficiency in these patients, manifested even before a decline in CD4+ lymphocyte counts is observed. B cell dysfunction with impaired antibody synthesis is the major immunologic characteristic of CVI patients. However, the immunologic abnormalities in CVI are not restricted to B cells, but often also involve T cell dysfunction, e.g. impaired proliferative response on stimulation. The B cells in CVI patients are not necessarily intrinsically defective, and impaired T cell “help” may be of importance for the B cell defect in these patients. T cell dysfunction may also be of importance for certain clinical manifestations in these patients not necessarily related to defective antibody production, e.g. increased incidence of granulomata and malignancies.
Current therapies. Antiretroviral therapy is the main component in the treatment of HIV-infected patients. However, although potent antiretroviral combination therapy may markedly increase the CD4+ and CD8+ lymphocyte counts in HIV-infected patients, impaired T cell function seems to persist, as indicated in the observations made in Example 1, tables I and 2B. Thus, there is a need for immunomodulating, agents in addition to antiretroviral therapy in these patients. Immunoglobulin substitution is the main component in the treatment of CVI patients. However, this substitution therapy does not restore the defective T and B cell function. Furthermore, in some clinical complications, e.g. noncaseating granulomata and persistent viral infections, there is need for therapy which more directly may exchange T cell function.
Therapeutic potential of novel drugs targeting T cell dysfunction. Although impaired T cell function is a well recognized immunologic feature of both HIV infection and CVI, the exact molecular mechanism for this T cell impairment is unknown. Therapeutical modalities directed against such intracellular defects may be of major importance in the treatment of these patients, and may have the potential to restore important immunologic defects in HIV-infected patients and in patients with CVI.
Hofmann et al. (Aids, Vol 7; 659–664, 1993) and WO 9-3/19766 has demonstrated that HIV-seropositive individuals without AIDS showed significant increase in intracellular cAMP levels and PKA activity in crude peripheral blood mononuclear cells (PBMC) from HIV-seropositive subjects. Examination of T cells was reported as data not shown and did not reach significance because of larger variability, probably induced by the T cell purification method. Their study further indicated that adenosine analogues such as 2′,5′ dideoxyadenosine (ddAdo) reduced cellular cAMP levels in PBMC and increased the cell proliferation. This effect was, however, concentration dependent such that concentrations in the range of 6 ng/ml were effective and higher concentrations were suppressive or did not further inhibit cAMP levels. It was not demonstrated similar effects in the T cells sampled from HIV-patients, neither a simple concentration/response relationship. They have used purified T cells in their examples but these cells were sampled from healthy blood donors and purified by positive selection that may lead to premature T cell activation.
Chu-Chung, Y S, Genieser H and Jastorff, B (WO 93)/21929-A) have shown treatment applied to cancer cells by antagonising cAMP-dependent protein kinases, by using phosphorothoate derivatives of cAMP.
In summary it is not known in the prior art the role of the cAMP level in T cells regarding cell proliferation and immune response during physiological conditions. Furthermore, it is not known the role of the protein kinase isoenzymes regarding T cell functions during physiological conditions and if cAMP/PKAI has immunomodulating activity. Although the results of Hofman et al (vide supra) suggested increased cAMP level in mononuclear cells, it was not presented documentation supporting a similar condition in T cells. It is thus not known if the cAMP level is increased in purified T cells and negatively selected (not activated) T cells from HIV+ subjects, and nothing regarding the cAMP/PKA pathway in patient suffering from CVI.